DNA Barcoding: An Emerging Global Standard for Species Identification

1. DNA Barcoding: An Emerging Global Standard for Species Identification Consortium for the Barcode of Life National Museum of Natural History Smithsonian Institution http://www.barcoding.si.edu 202/633-0808; fax 202/633-2938

2. A DNA barcode is a short gene sequence taken from standardized portions of the genome, used to identify species

3. D-Loop Small ribosomal RNA Large ribosomal RNA Cyt b ND1 ND6 COI COI ND5 L-strand ND2 H-strand ND4 COI ND4L COII ND3 ATPase subunit 8 COIII ATPase subunit 6 The Mitochondrial Genome

4. Uses of DNA Barcodes Applied tool for identifying regulated species: • Disease vectors, agricultural pests, invasives • Environmental indicators, protected species • Using minimal samples, damaged specimens, gut contents, droppings Research tool for improving species-level taxonomy: • Associating all life history stages, genders • Testing species boundaries, finding new variants “Triage” tool for flagging potential new species: • Undescribed and cryptic species

5. Using DNA Barcodes • Establish reference library of barcodes from identified voucher specimens • If necessary, revise species limits • Then: • Identify unknowns by searching against reference sequences • Look for matches (mismatches) against ‘library on a chip’ • Before long: Analyze relative abundance in multi-species samples

6. Reactions to Barcoding: 2004 • From ecologists and other users:“This is what we need! How soon can we get started?” • From traditional taxonomists:“Species should be based on lots of characters, not just barcodes” • From forward-looking taxonomists:“Using molecular data as species diagnostics isn’t new, but standardization and broad implementation are great!” • From barcoding practitioners:“I had my doubts at the beginning, but it really works as a tool for identification (96% accurate in a recent mollusc paper) and it is at least as good as traditional approaches to discovering new species.”

7. What DNA Barcoding is NOT • Barcoding is not DNA taxonomy; no single gene (or character) is adequate • Barcoding is not Tree of Life; barcode clusters are not phylogenetic trees • Barcoding is not just COI; standardizing on one region has benefits and limits • Molecules in taxonomy is not new; but large-scale and standardization are new • Barcoding can help to create a 21st century research environment for taxonomy

10. What DNA Barcoding is NOT • Barcoding is not DNA taxonomy; no single gene (or character) is adequate • Barcoding is not Tree of Life; barcode clusters are not phylogenetic trees • Barcoding is not just COI; standardizing on one region has benefits and limits • Molecules in taxonomy is not new; but large-scale and standardization are new • BUT…Barcoding can help to create a 21st century research environment for taxonomy

11. BARCODE Data Standard Specimen Metadata Voucher Specimen Species Name GeoreferenceHabitatCharacter setsImagesBehaviorOther genes Indices - Catalog of Life - GBIF/ECAT Nomenclators - Zoo Record - IPNI NameBank Publication links - New species Barcode Sequence Trace files Primers Other Databases Literature(link to content or citation) PhylogeneticPop’n GeneticsEcological

12. Current Norm: High throughput Large capacity PCR and sequencing reactions ABI 3100 capillary automated sequencer

13. Cost of Reagents and Disposables

14. Producing Barcode Data: 2008 Faster, more portable: Hundreds of samples per hour Integrated DNA microchips Table-top microfluidic systems

15. Reference vs. Micro-Barcodes • BARCODE reference records • Adhere to data standards • Bidirectional reads, 500+ bp long • Linked to voucher, species name • Query barcode records • Used in BLAST or other searches • Often single pass reads • Often very short – 100+ bp for good IDs • Can cost less than $2, take less than 6 hours

16. Producing Barcode Data: 2010?Barcode data anywhere, instantly • Data in seconds to minutes • Pennies per sample • Link to reference database • A taxonomic GPS • Usable by non-specialists

17. Consortium for the Barcode of Life (CBOL) • First barcoding publications in 2002 • Cold Spring Harbor planning workshops in 2003 • Sloan Foundation grant, launch in May 2004 • Secretariat opens at Smithsonian, September 2004 • First international conference February 2005 • Now an international affiliation of: • 130+ Members Org’s, 40 countries, 6 continents • Natural history museums, biodiversity organizations • Users: e.g., government agencies • Private sector biotech companies, database providers

18. CBOL Member Organizations June 2006: 120 Member Organizations, 40 countries

19. CBOL’s Underlying Principles • Standardization, cost-effectiveness • Minimalism (scientific) – short sequence • Global participation • Tangible, realistic goals, near-term results • Respond to applied user communities • Minimalisim (organizational) – Coordination/Facilitation of Bottom-Up activities, build on existing activities, avoid new ones

20. CBOL’s Working Groups • Database: Designing/constructing the Barcode Section of GenBank • DNA: Protocols for formalin-fixed and old museum specimens; advice to new labs • Data Analysis: Beyond phenetic methods; population genetics perspective • Plants: Identify gene region(s) for barcoding

21. Outreach to Developing Countries • Regional meetings in: • Cape Town, South Africa, 7-8 April 2006, SANBI • Nairobi, Kenya, 18-19 October 2006, NMK • Sao Paolo, Brazil, February 2007, INPA • Southern Asia, 2nd quarter 2007 • Second International Barcode Conference • Singapore, 13-15 June 2007 • Support from CBOL, host governments and international development agencies

22. Goals of Regional Meetings • Raise awareness • Explore potential applications in the region • Assess greatest needs and opportunities in the region • Identify highest priorities, construct national and regional action plans • Start intra-regional networks and intercontinental partnerships

23. Current and Planned Projects • Four Working Groups • FishBOL and All Birds Initiatives • Developing “Demonstrator Systems by 2008 • African Scale Insect Barcoding Initiative (planned at Cape Town Regional Meeting) • International Network for Barcoding Invasive and Pest Species (INBIPS) • Forming a Conservation Committee

24. Demonstrator Development GOAL: By June 2008, create an operational system for global ID of an important group • Mosquitoes (disease vectors) • Barcode 85% (2932) of 3449 known species • Minimum 5 specimens per species • Reliance on archival collections • Tephritid fruit flies (agricultural pests) • Barcode 2000 species of 4500, 5 per species • All pests and beneficials, with closest relatives • Invasive non-destructive sampling of existing collections

25. ABBI and FISH-BOL • Global initiatives to create reference library • Enable users to adopt barcode ID systems • All-species barcode database will: • Strengthen specimen/species data • Improve collections, tissue/DNA resources • Attract users to barcoding for specimen IDs • Regional Working Groups • Small Steering Committee and CBOL

26. BoLD Data System • Developed/hosted by Univ. Guelph • Workbench for most barcode projects • Laboratory Information Management System (LIMS) for assembling data • Management and Analysis System • Identification system for matching unknowns to reference records • Uploading to GenBank

28. IDS – Identification System

29. Linkage to Mapping System

30. Launching CBOL Projects Assembling Steering Committee • Users • Taxonomists, collection curators • Service providers (BoLD, analytical labs) • Plan for scope, timetable, logistics • Pilot tests of primers, PCR amplification • Assemble pipeline of specimens to lab

31. DNA from identified voucher Create BARCODE reference record Refine taxonomy of group ID unknowns DNA from unidentified immature specimen Create BARCODE reference records Associate immatures with names DNA from identified adult voucher Repository of provisional vouchers ID unknowns Add names to vouchered immatures Refine taxonomy of group